Protein production method, fusion protein, and antiserum

ABSTRACT

Disclosed are a highly efficient method for production of heterologous proteins performed by utilizing microorganisms, as well as fusion proteins, and an antiserum. The method includes a method for production of a protein (A) in the form of a fusion protein, comprising the steps of (a) preparing a DNA which codes for a fusion protein comprising the peptide chain forming the protein (A) and the C-terminal peptide or its fragment (B) of the Cry proteins produced by  Bacillus thuringiensis , and (b) introducing the DNA into a host bacterium to transform the same, and (c) allowing the fusion protein to be expressed in the transformed host bacterium, as well as a method for production of the protein (A) itself comprising a further step of removing the peptide chain (B) from the fusion protein obtained.

TECHNICAL FIELD

The present invention relates to a method for production of proteins, inparticular to a method for production of heterologous proteins usingbacteria as a host, and more specifically a highly efficient method forproduction of heterologous proteins performed by utilizing thecharacteristic properties of Cry proteins, the proteins formed byBacillus thuringiensis. The present invention further relates to fusionproteins produced by the method, antiserum and antibodies to such fusionproteins, and reagents containing them, as well as a method foranalysis.

BACKGROUND ART

As for production of proteins/enzymes, i.e., major components of variousbiological products, simple, easy and low-cost methods for theirproduction have been sought, such as introduction of their genes, ifthey are isolated, into microorganisms, like E. coli, and letting theproteins/enzymes be expressed and accumulate in them. However, there arealso many cases in which attempts fail to let a heterologous proteinexpress/accumulate in the cells of microorganisms. The cause of suchfailures becomes particularly notable when higher production efficiencyis sought. That is, the heterologous protein biosynthesized in the cellsof a microorganism, which accumulates forming insoluble inclusionbodies, gets inactivated in the process. Generally, it is very difficultto solubilize such insoluble inclusion bodies of a protein and restoringthe biological activity of the protein.

Further, in the case where the heterologous protein to be produced has acytotoxicity, accumulation of that toxic protein adversely affects theproliferation/survival of the very host cells, resulting in loweredproduction yield, and further, in death of the host cells.

Due to these drawbacks, production of proteins/enzymes, which are majorcomponents of various biological products, often has to rely ontime-consuming and costly methods, such as utilizing the livingorganisms that intrinsically produce the protein/enzyme. There seems tobe not a small number of cases in which these drawbacks form a factorthat hindering development of an efficient method of their industrialproduction, and this is one of the problems to be solved.

On the other hand, insecticidal proteins produced by an aerobic soilbacterium, Bacillus thuringiensis, has long been known (see as a reviewe.g., Non-patent Document 1). During its sporulation, Bacillusthuringiensis also produces, separately from spores, generally a singlelarge parasporal inclusion body consisting mainly of a crystal protein(this is called “Cry protein”). Cry proteins consist of about 1000-1200amino acids, and many of them are known to be produced by variousBacillus thuringiensis strains. About half of them have been found tohave an insecticidal activity specific to certain insects, and some ofthose strains which produce a Cry protein having such an activity havebeen widely used as BT insecticides. When ingested by larvae ofrespective target insects, each of those Cry proteins which have aninsecticidal activity undergoes cleavage by the digestive fluid at apredetermined position of its amino acid sequence and thus a peptide onits C-terminus side (consisting about 400-500 amino acids: abbreviatedto “Cter”) is removed, leaving behind a peptide on the N-terminus side(NB: a short peptide at the N-terminus is also removed), which exhibitsa potent insecticidal activity. For example, in the case of a proteinnamed Cry4Aa, an N-terminus region consisting of Met 1 to Gln695 (inparticular, a peptide left behind after Met1 to Try57 are removed) ofits entire amino acid sequence acts as the very insecticidal componenton its target insects (Culex pipiens pallens and the like). Its Cter,which consists of an amino acid sequence starting with Ile696 and isdeleted by cleavage, is unnecessary part for the insecticidal activity(see Non-Patent Document 2). And, as for a protein known as Cry1Aa,which is an insecticidal specific to lepidopteran insects (butterfliesand moths), the region consisting of Met1 to Lys621 on the N terminalside of its amino acid sequence (in particular, the part of the regionleft behind after further removal of Met1 to Arg28) works as the veryinsecticidal component, and its Cter, which consists of an amino acidsequence starting with Ala622, is deleted by cleavage (Non-PatentDocument 3). Furthermore, as to Cry1Ac, which is also an insecticidaltoxin specific to Lepidopteran insects (butterflies and moths) likeCry1Aa, its very insecticidal component consists of Met1 to Lys623 (inparticular, the part of the region left behind after removal of Met1 toArg28) of its amino acid sequence, and its Cter, which is located on theC-terminal side and consists of amino acid sequence starting withAla624, does not take part in the generation of insecticidal activity,but is removed by cleavage by the action of the digestive fluid in thelarvae (Non-Patent Document 4). Further, in recent years, Cry proteinshave been found with which no insecticidal activity is as yet known, andsome of them are found to specifically destroy human cancer cells, somespecifically kill Tricomonas vaginalis, the pathogen protozoa causinghuman tricomonasis, and some exhibit a potent worm-killing activity toNematoda. Though they differ in their biological activity and in theirtargets at which they exhibit toxic effects, the Cry proteins have acommon characteristic property that they occur within Bacillusthuringiensis cells in the form of a large parasporal inclusion body.

-   [Non-Patent Document 1] M. Ohba, H. Hori and H. Sakai, “Bacillus    thuringiensis: Science of Insecticidal Proteins”, Industrial    Publishing & Consulting, Inc., Feb. 28, 2005.-   [Non-Patent Document 2] M. Yamagiwa et al., Appl. Environ.    Microbiol. 65: 3464-3469 (1999)-   [Non-Patent Document 3] P. Grochulski et al., J. Mol. Biol. 254:    447-464 (1995)-   [Non-Patent Document 4] J. N. Aronson and H. C. Arvidson, Appl.    Environ. Microbiol. 53: 416-421 (1999)

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

Against the above background, the objective of the present invention isto provide a method for production of heterologous proteins/enzymesutilizing microorganisms, with improved efficiency compared withconventional methods.

Means to Solve the Problem

The present inventors found that when brought to express in hostbacterium cells, such as E. coli cells, a fusion protein consisting ofthe Cter of one of the Cry proteins and some other protein which wasfused with it, an insoluble inclusion bodies containing the fusionprotein (hereinafter referred to as a “crystal”) formed in the cells,and thus this heterologous protein accumulated in a great amount in thecells, and that those crystals had following excellent propertiesdistinguished from those of other insoluble inclusion bodies so farknown:

a. that the heterologous protein/enzyme has been contained in thecrystals, with its biological activities kept intact,

b. that crystals are efficiently solubilized in an alkaline buffersolution with a pH of 10-11,

c. that after solubilized, the heterologous protein/enzyme can be easilyrecovered in the supernatant, with its biological activities keptintact,

d. that even a cytotoxic heterologous protein can be efficientlyproduced through crystal formation, and

e. that crystals can be stored as they are for a long time, with theirstability being kept uncompromised.

The present inventors further found that the full-length Cter is notindispensable for such crystals to be formed, but a part consisting ofsome 140-160 amino acids on its N-terminal side suffices. In particular,examination was performed, based on 4AaCter(696-851) of Cry4Aa2, whichis a Cter's N-terminal ⅓-long fragment and with which it was firstdiscovered that a fusion protein consisting of it and some other proteinforms crystals, and by selecting fragments of various other Cter's aminoacid sequences according to their similarity to the former fragmentbased on the alignment technique. As a result, formation of similarcrystals was found with them. It was also found that an antiserum whichis created by immunizing an animal with a fusion protein obtainedaccording to the present invention is reactive with the originalprotein, i.e., the protein prior to fused with the Cter employed. Thepresent invention has been completed through further studies on thebasis of these findings.

Thus, the present invention provides what follows:

1. A method for production of a protein (A) in the form of a fusionprotein, comprising the steps of

(a) preparing a DNA which codes for a fusion protein comprising thepeptide chain forming the protein (A) and other peptide chain (B), onthe N- or C-terminal side of the latter the former being combined,wherein the peptide chain (B) is a C-terminal peptide chain included inthe amino acid sequence of one of the Cry proteins produced by Bacillusthuringiensis listed in Table 1 or 2 and including in itself acorresponding partial sequence identified in the tables,

TABLE 1 C-terminal peptide, and Cry proteins partial sequence (SEQ IDNO:) Cry1Aa1(SEQ ID NO: 50), Cry1Aa2(SEQ ID NO: 51), C-terminal peptide:the part starting Cry1Aa3(SEQ ID NO: 52), Cry1Aa4(SEQ ID NO: 53), fromAla622 Cry1Aa5(SEQ ID NO: 54), Cry1Aa8(SEQ ID NO: 55), Partial sequence:Ala622-Pro777 Cry1Aa9(SEQ ID NO: 56), Cry1Aa10(SEQ ID NO: 57), (SEQ IDNO: 1) Cry1Aa11(SEQ ID NO: 58), Cry1Aa12(SEQ ID NO: 59), Cry1Aa13(SEQ IDNO: 60), Cry1Aa14(SEQ ID NO: 61) Cry1Ab3(SEQ ID NO: 62), Cry1Ab4(SEQ IDNO: 63), C-terminal peptide: the part starting Cry1Ab8(SEQ ID NO: 64),Cry1Ab9(SEQ ID NO: 65), from Ala623 Cry1Ab10(SEQ ID NO: 66),Cry1Ab12(SEQ ID NO: 67), Partial sequence: Ala623-Pro778 Cry1Ab13(SEQ IDNO: 68), Cry1Ab15(SEQ ID NO: 69), (SEQ ID NO: 2) Cry1Ab16(SEQ ID NO:70), Cry1Ab17(SEQ ID NO: 71), Cry1Ab21(SEQ ID NO: 72) Cry1Ab2(SEQ ID NO:73) C-terminal peptide: the part starting from Ala624 Partial sequence:Ala624-Pro779 (SEQ ID NO: 3) Cry1Ac1(SEQ ID NO: 74), Cry1Ac4(SEQ ID NO:75), C-terminal peptide: the part starting Cry1Ac7(SEQ ID NO: 76),Cry1Ac8(SEQ ID NO: 77), from Ala624 Cry1Ac9(SEQ ID NO: 78), Cry1Ac10(SEQID NO: 79), Partial sequence: Ala624-Pro779 Cry1Ac11(SEQ ID NO: 80),Cry1Ac16(SEQ ID NO: 81), (SEQ ID NO: 4) Cry1Ac19(SEQ ID NO: 82)Cry1Ac5(SEQ ID NO: 83), Cry1Ac12(SEQ ID NO: 84), C-terminal peptide: thepart starting Cry1Ac14(SEQ ID NO: 85), Cry1Ac15(SEQ ID NO: 86), fromAla623 Cry1Ac20(SEQ ID NO: 87) Partial sequence: Ala623-Pro778 (SEQ IDNO: 5)

TABLE 2 C-terminal peptide, and Cry proteins Partial sequence(SEQ IDNO:) Cry4Aa1(SEQ ID NO: 88), C-terminal peptide: the part Cry4Aa2(SEQ IDNO: 89), starting from Ile696 Cry4Aa3(SEQ ID NO: 90) Partial sequence:Ile696-Pro851 (SEQ ID NO: 6) or Ile801-Ser829 (SEQ ID NO: 7) Cry4Ba1(SEQID NO: 91), C-terminal peptide: the part Cry4Ba2(SEQ ID NO: 92),starting from Val652 Cry4Ba5(SEQ ID NO: 93) Partial sequence:Val652-Pro807 (SEQ ID NO: 8) or Ile757-Ser785 (SEQ ID NO: 9) Cry4Ba4(SEQID NO: 94) C-terminal peptide: the part starting from Val651 Partialsequence: Val651-Pro806 (SEQ ID NO: 10) or Ile756-Ser784 (SEQ ID NO: 11)Cry4Ba3(SEQ ID NO: 95) C-terminal peptide: the part starting from Val652Partial sequence: Val652-Pro807 (SEQ ID NO: 12) or Ile756-Ser784 (SEQ IDNO: 13) Cry8Ca1(SEQ ID NO: 96) C-terminal peptide: the part startingfrom Lys672 Partial sequence: Lys672-Pro829 (SEQ ID NO: 14)

(b) introducing the DNA into a host bacterium to transform the same, and

(c) allowing the fusion protein to be expressed in the host bacteriumwhich has been transformed.

2. The method for production according to 1 above, wherein in the stepof preparing the DNA which codes for a fusion protein comprising thepeptide chain forming the protein (A) and the other peptide chain (B),on the N- or C-terminal side of the latter the former being combined, aDNA coding for an amino acid sequence which provides a specific cleavagesite for a proteolytic enzyme is interposed between the DNA which codesfor the peptide chain forming protein (A) and the DNA which codes forthe peptide chain (B).

3. The method for production according to 1 or 2 above comprising afurther step of fracturing the host bacterium containing the fusionprotein thus expressed to collect the fusion protein.

4. The method for production according to 3 above comprising a furtherstep of purifying the collected fusion protein through solubilization ofthe same in an alkaline aqueous solution.

5. The method for production of the protein (A) comprising a step ofremoving the peptide chain (B) from the fusion protein obtainedaccording to 4 above.

6. The method for production according to 5 above, wherein the removalof the peptide chain (B) is done by treating a specific cleavage sitefor a proteolytic enzyme, which the fusion protein has between thepeptide chain forming the protein (A) and peptide chain (B), with theproteolytic enzyme.

7. A fusion protein produced by the method according to 3 or 4 above.

8. An antiserum reactive to the protein (A) which antiserum is obtainedby immunizing a mammalian animal with the fusion protein according to 7above, and then collecting the serum of the animal.

9. The antiserum according to 7 or 8 above, wherein the protein (A) isC-reactive protein.

10. An antibody to the protein (A) isolated from the antiserum accordingto 8 or 9 above.

11. A testing reagent comprising the antiserum according to 8 or 9above.

12. A testing reagent comprising the antibody according to 10 above.

13. A method for analysis of a sample for protein (A), comprising thesteps of bringing the sample into contact with the antiserum accordingto 8 above, or the antibody according to 9 above, to form anantigen-antibody complex, and detecting the antigen-antibody complex.

14. The method according to 13 above, wherein the protein (A) isC-reactive protein.

Effect of the Invention

According to the present invention, utilizing a bacterium, such as E.coli, as a host, a heterologous protein can be produced in the form of afusion protein in a great amount within the cells of the bacterium, inthe form of insoluble crystals, maintaining (but in a potential manner)activities of the original protein. As these insoluble crystals, in thesituation where they are, do not exhibit the activities of the originalheterologous protein in the host bacterium, even proteins otherwiseharmful or lethal to the host can be produced in a large scale.Furthermore, the crystals can be easily isolated, and then solubilizedin an alkaline condition, collected and purified. The solubilized fusionprotein has the activities of the original, heterologous protein, andfurther it is also possible to readily recover the original heterologousprotein through removal, by any of proper methods, of the portion whichhas been derived from the Cter employed. Therefore, the presentinvention enables production of heterologous proteins utilizing abacterium, such as E. coli, as a host, with dramatically improvedefficiency.

Furthermore, the present invention also enables to obtain, in a largeamount, even such proteins as have been obtainable so far only in atrace amount, in the form in which the proteins are fused to aC-terminal peptide chain of a Cry protein. Thus, it makes it easier toobtain antisera to original proteins, through immunization of mammaliananimals (e.g., rabbit, goat) with their fusion proteins and collectionof the sera. Such antisera can be used, e.g., as testing reagents, fordetection or measurement of the original proteins in given samples(e.g., a biological sample such as body tissues, blood, plasma, orserum). Further, antibodies (polyclonal antibodies) to the originalproteins can also be isolated from such antisera, and can be used forthe same purpose.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the primer sets used for preparation of syntheticDNAs which then were used for construction of the gene coding for4AaCter (696-851) by repetitive PCR.

FIG. 2 is a schematic illustration of the process for construction ofthe DNA coding for the entire 4AaCter(696-851) by repetitive PCR.

FIG. 3 illustrates the order of combination of GST and 4AaCter (696-851)in their fusion protein.

FIG. 4 is a schematic diagram illustrating the flow of the steps fromthe construction of the DNA coding for 4AaCter (696-851) up to theconstruction of an expression vector for the fusion protein.

FIG. 5 illustrates the primer pair which was employed to amplify the DNAfragment coding for 4AaCter(696-851).

FIG. 6 illustrates a map of expression vector pGEX-6P-1.

FIG. 7 is a photograph showing crystal formation in E. coli wells whichwere transformed with pGST-4AaCter and induced for expression of it.

FIG. 8 is a photograph showing localization of crystals in the insolublefraction in E. coli cells which were transformed with pGST-4AaCter andinduced for expression of it. In the figure, M: size marker, 1: wholecell following induction of expression, 2: supernatant followingfracturing the cells, 3: precipitate following fracturing the cells, 4:supernatant following solubilization.

FIG. 9 is a diagram showing the results of examination for crystalforming property of fusion proteins which were produced using variousfragments of Cter from Cry4Aa.

FIG. 10 is a diagram showing the two different orders of combination ofMM29 kD and 4AaCter (696-851) in their fusion proteins.

FIG. 11 is a schematic diagram illustrating the flow of the steps ofconstruction of the expression vector for production of 4AaCter-MM29 kD.

FIG. 12 illustrates a map of pGEX-4T-3.

FIG. 13 illustrates the primer pair which was employed to remove theentire open reading frame (ORF) of GST except “ATG”.

FIG. 14 is a diagram showing the nucleotide sequence near the site where“ATG” is combined with the 5′-end of the DNA coding for MM29 kD.

FIG. 15 is a schematic diagram illustrating the flow of construction ofthe expression vector for production of MM29 kD-4AaCter.

FIG. 16 presents photographs showing crystal formation in the E. colicells which were transformed with pΔGST-4AaCter-MM29 kD or pΔGST-MM29kD-4AaCter, respectively, and induced for their expression.

FIG. 17 present photographs showing the results of SDA-PAGE performedwith 4AaCter-MM29 kD and MM29 kD-4AaCter.

FIG. 18 is a schematic diagram illustrating the flow of construction ofexpression vector pGST-1AaCter.

FIG. 19 presents photographs showing crystal formation in E. coli cellswhich were transformed with pGST-1AaCter and induced for expression ofit.

FIG. 20 presents photographs showing the result of SDS-PAGEdemonstrating the insoluble-fraction localization of crystals obtainedfrom the E. coli cells which were transformed with pGST-1AaCter andinduced for its expression. In the figure, M: size marker, 1: IPTG(−)total proteins, 2: IPTG(+), 3: IPTG(+) centrifugation supernatant(soluble protein fraction), 4: IPTG(+) centrifugation precipitate(insoluble protein fraction).

FIG. 21 presents photographs showing the result of SDA-PAGE carried outfollowing alkaline solubilization of the crystals obtained from E. colicells were transformed with pGST-1AaCter and induced for expression. Inthe figure, M: size marker, 1: centrifugation supernatant (solubleprotein fraction), 2: centrifugation precipitate (insoluble proteinfraction).

FIG. 22 presents photographs showing crystal formation in E. coli cellswhich were transformed with pGST-1AcCter and induced for expression.

FIG. 23 present photographs showing the result of SDS-PAGE demonstratingthe insoluble-fraction localization of crystals obtained from E. colicells which were transformed with pGST-1AcCter and induced forexpression. In the figure, M: size marker, 1: IPTG(−) total proteins, 2:IPTG(+) total proteins, 3: IPTG(+) entrifugation precipitate (insolubleprotein fraction), 4: IPTG(+) centrifugation supernatant (solubleprotein fraction).

FIG. 24 presents photographs showing the results of SDA-PAGE followingalkaline solubilization of crystals obtained from E. coli cells whichwere transformed with pGST-1AcCter and induced for expression,indicating the result of solubility test of the crystals formed with1AcCter. In the figure, M: size marker, 1: centrifugation precipitate(insoluble protein fraction), 2: centrifugation supernatant (solubleprotein fraction).

FIG. 25 is a photograph showing the result of SDS-PAGE for confirmationof expression of CRP and 4AaCter-CRP.

FIG. 26 is a photograph showing the result of Western blotting of4AaCter-CRP and human-derived CRP.

FIG. 27 is a photograph showing the result of Western blotting of4AaCter and 4AaCter-CRP.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present specification, amino acid numbers are determined byassigning “1” to the methionine (Met) residue corresponds to the startcodon in the original Cry protein.

The ability of a fusion protein to form crystals is thought to be theproperty that is common to the Cter region of the Cry proteins, whichare characterized by formation of a large parasporal inclusion body, foras shown in the Examination section, crystal formation was alikeconfirmed in the experiments employing combinations of different Cter'sand different heterologous proteins, and since the structure of Cter'sare highly conserved among insecticidal toxins.

A large number of Cry proteins are known (Bacillus thuringiensis ToxinNomenclature: http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/).Of the full-length amino acid sequence of a Cry protein, the N-terminalside portion which determines such biological activities as aninsecticidal activity, is not needed for the purpose of the presentinvention, and therefore the remaining portion, the C-terminal peptide(Cter), may be utilized for formation of fusion proteins with theprotein of interest. Further, in utilizing a Cter, full length of it isnot necessary, but its N-terminal part consisting of about 140-160 aminoacids suffices, and its fragments may also be used.

As to Cry4Aa1, Cry4Aa2 and Cry4Aa3 (Cry4Aa group), for example, whichhave in common the identical partial sequence Ile696-Pro851 (SEQ IDNO:6) inside the Cter, the portion starting from Ile696, it has beenconfirmed that a fusion protein consisting of the peptide having thispartial sequence and a heterologous protein forms crystals (cf.Examples). Further, among various other partial sequences includedinside this partial sequence, the sequence Ile801-Ser829 (SEQ ID NO:7)and some other partial sequences including this within themselves alsoprovided peptides which gave, in combination with a heterologousprotein, such a fusion proteins that formed crystals, whereas partialsequences including no Ile801-Ser829 within themselves failed to formcrystals (Examples). Therefore, Ile801-Ser829 (SEQ ID NO:7) isconsidered to be the very sequence that confers fusion proteins theability to form crystals. Thus, Cter's partial sequences which includethis sequence in themselves can be used to form fusion proteins withheterologous proteins so as to confer the latter the ability to formcrystals.

Furthermore, Cry4Ba1, Cry4Ba2 and Cry4Ba5 share a completely identicalpartial amino acid sequence Val652-Pro807 (SEQ ID NO:8), whichcorresponds to the above-mentioned Cter fragment Ile696-Pro851 ofCry4Aa1, and, further, all of them include a sequence (Ile757-Ser785,SEQ ID NO:9) within themselves which is completely identical toIle801-Ser829 (SEQ ID NO:7) of Cry4Aa1. Therefore, as to Cry4Ba1,Cry4Ba2 and Cry4Ba5, either their Cter themselves (the portion startingfrom Val652) or their Cter's partial sequences which includeIle757-Ser785 within themselves can be used as desired for the purposeof the present invention.

As to Cry4Ba4, a partial sequence Val651-Pro806 (SEQ ID NO:10) withinits Cter (the portion stating from Val651), which corresponds to theabove-mentioned Cter fragment Ile696-Pro851 of Cry4Aa1, is completelyidentical to the partial amino acid sequence Val652-Pro807 of Cry4Ba1,Cry4Ba2 and Cry4Ba5, and they differ only in their starting region intheir respective Cry proteins. Therefore, again, Cry4Ba4 includes withinitself a sequence (Ile756-Ser784, SEQ ID NO:11) that is the same asIle801-Ser829 (SEQ ID NO:7) of Cry4Aa1. Thus, as to Cry4Ba4 also, eitherits Cter itself (the portion starting from Val651) or any of itsfragments which includes Ile756-Ser784 within itself can be used asdesired for the purpose of the present invention.

As to Cry4Ba3, a partial sequence Val652-Pro807 (SEQ ID NO:12) withinits Cter (the portion starting from Val652), which corresponds to theabove-mentioned Cter fragment le696-Pro851 of Cry4Aa1, also includeswithin itself a sequence (Ile757-Ser785, SEQ ID NO: 13) which iscompletely identical to Ile801-Ser829 (SEQ ID NO:7) of Cry4Aa1.Therefore, as to Cry4Ba3, too, either its Cter (the portion startingfrom Val652) itself or any of its fragments which includes Ile757-Ser785within itself can be used as desired for the purpose of the presentinvention.

As for the Cry1Aa group including e.g., Cry1Aa1 as shown in the abovetable, their Cter's are the portions all starting from Ala622, amongwhich their partial sequences Ala622-Pro777 are completely identicalwith one another in their amino acids. A fusion protein of the peptideconsisting of this identical portion and a heterologous protein wasconfirmed to form crystals (see Example in which a peptide originatingfrom Cry1Aa3 is utilized). Therefore, as regards the Cry1Aa group,either their Cter's (the portion starting from Ala622) themselves or anyof their fragments which includes Ala622-Pro777 within itself can beused as desired in the present invention.

Regarding the CryAb group including e.g., Cry1Ab3 (but except Cry1Ab2)as shown in the above table, their Cter's are the portions all startingfrom Ala623, among which their partial sequence Ala623-Pro778, whichcorrespond to the above-mentioned Cter fragment Ile696-Pro851 ofCry4Aa1, are completely identical with one another in their amino acids.And this partial sequence, when compared with the partial sequenceAla622-Pro777 of the Cter's of the above Cry1Aa group, differs only inthat the Gln664 in the Cry1Aa group is replaced with 665Lys in the CryAbgroup, and has no other difference except that the amino acid positionnumbers in the former partial sequence as a whole are greater by 1 thanthe latter. As the proteins belonging to the CryAb group are those whichform a parasporal inclusion bodies and the partial sequenceAla623-Pro778 or their Cter's differ only in one single amino acidcompared with the partial sequence Ala622-Pro777 of the Cter of theCry1Aa group (with which formation of crystals is confirmed in Example),either the Cter's of the CryAb group (the portion starting from Ala623)themselves or any of their fragments which include Ala623-Pro778 withinthemselves can be used as desired in the present invention.

The Cter of Cry1Ab2 is the portion which starts from Ala624, and anamino acid sequence Ala624-Pro779 included within it differs only inthat Ser703 and Asp712 of the Cry1Ab group are replaced in it withAsn704 and His713, respectively, and has no other difference except thatits amino acid position numbers as a whole are shifted by 1. For thisreason, and since Cry1Ab2 is a protein which forms parasporal inclusionbodies, either the Cter of this (the portion starting from Ala624)itself or any of its fragments which include Ala624-Pro779 withinthemselves can be used as desired in the present invention.

In the above tables, the group consisting of Cry1Ac1, Cry1Ac4, Cry1Ac7,Cry1Ac8, Cry1Ac9, Cry1Ac10, Cry1Ac11, Cry1Ac16 and Cry1Ac19 (Cry1Ac(I)group) have their Cter's which start from Ala624, and the groupconsisting of Cry1Ac5, Cry1Ac12, Cry1Ac14, Cry1Ac15, and Cry1Ac20(Cry1Ac(II) group) have their Cter's which start from Ala623. And thepartial sequence Ala624-Pro779 of the former is completely identical intheir amino acids to the partial sequence Ala623-Pro778 of the latter.Judging from the fact that a fusion protein of the peptide consisting ofthe partial sequence Ala624-Pro779 of Cry1Ac1 and a heterologous proteinwas confirmed to form crystals (see Examples), also with the Cry1Ac(I)and Cry1Ac(II) groups, either their Cter's themselves or any of thefragments of the Cter which include the partial sequence Ala624-Pro779for the Cry1Ac(I) group or the partial sequence Ala623-Pro778 for theCry1Ac(II) group, can be used in the present invention.

In the present invention, the DNA coding for the fusion proteinconsisting a protein of interest and a Cter (or its fragment) isprepared by combining a DNA coding for the Cter or its fragmentmentioned above is combined in-frame with a DNA coding for the proteinof interest on its 3′ or 5′ end.

In the present invention, by employing as a template the total DNA of aBacillus thuringiensis strain which has the gene of a particular Cryprotein that is to be utilized, a DNA coding for the Cter (or itsfragment) of the protein can be prepared by PCR. In this preparationprocess, the primers employed may be provided with restriction sites bya conventional method for incorporation into an expression vector. Inthe case where the template total DNA of the Bacillus thuringiensisstrain needed is unavailable, the DNA region of interest may besynthesized by first preparing primer DNAs (50-60 basis at longest),several to about 10 in number, that cover the entire region of the Cter(or its fragment) by a conventional method based onpublicized/registered (e.g., at DDBJ) information of that gene'snucleotide sequence/amino acid sequences, and then carrying out arecursive PCR using the primer DNAs.

The technique of recursive PCR is well known to those skilled in theart. Synthesis of the DNA by this method for the region of Cter (or itsfragment) may be carried out in the following manner, by way of example,4AaCter(696-851) [Ile696-Pro851 in the Cry4Aa group]: the nucleotidesequence coding for 4AaCter(696-851) is divided into 10 portions andeach portion is chemically synthesized as primers (FIG. 1), in such amanner as leaving complementary bases at its end(s) for permitting itshybridization with adjacent portion(s) at their ends, and using these, arecursive PCR is carried out to obtain the DNA coding for thefull-length 4AaCter (696-851). In the nucleotide sequences presented inFIG. 1, the bases shown by upper-case letters are those located withinthe nucleotide sequence coding for 4AaCter (696-851), and the basesshown by lower-case letters are those which were added for conveniencein manipulation.

(1) Primer C1-1-f (SEQ ID NO:15)

(2) Primer C1-1&2-r (SEQ ID NO:16)

(3) Primer C1-2&3-f (SEQ ID NO:17)

(4) Primer C1-3&4-r (SEQ ID NO:18)

(5) Primer C1-4&5-f (SEQ ID NO:19)

(6) Primer C1-5&6-r (SEQ ID NO:20)

(7) Primer C1-6&7-f (SEQ ID NO:21)

(8) Primer C1-7&8-r (SEQ ID NO:22)

(9) Primer C1-8&9-f (SEQ ID NO:23)

(10) Primer C1-9-r (SEQ ID NO:24)

A DNA fragment prepared by PCR using a pair of primers C1-1-f andC1-1&2r, which had at one end of them sequences complementary with eachother, and a DNA fragment having at both ends respective complementarysequences and prepared by PCR using a series of primer pairs, C1-2&3-f,C1-3&4-r, C1-4&5f, C1-5&6-r, C1-6&7-f and C1-7&8-r, and a DNA fragmentprepared by PCR using a pair of primers C1-8&9-f and C1-9r wereprovided. Using these fragments, the DNA (SEQ ID NO:25) coding for thefull-length 4AaCter(696-851) is prepared by PCR (FIG. 2).

It is also possible, in order to introduce a spacer which gives an aminoacid sequence that can be cleaved specifically by an enzyme (e.g., anamino acid sequence targeted by a protease) between the heterologousprotein of interest and the Cter (or its fragment), to introduce inadvance a DNA coding for such a spacer. Insertion of such a DNA can bedone by choosing a proper nucleotide sequence for the primers, which isalso well known to those skilled in the art.

When the gene prepared as above is introduced into a host bacterium,such as E. coli, and the host is induced to express the fusion protein,the fusion protein accumulates within the host cells in a great amountforming crystals. Crystals thus formed can be readily observed under anoptical microscope.

Through fracturing the host cells obtained above which have a largeamount of accumulated crystals, and centrifugation which follows, thecrystals can be collected as the precipitate. The crystals thuscollected can be dissolved by suspending them in an alkaline aqueoussolution, e.g., sodium a carbonate buffer with a pH of about 10.5-12 andincubating the mixture at 37° C. for 30 minutes to 2 hours. Afterdissolution of the crystals, the fusion protein, which retains thebiological activity is recovered in the supernatant by centrifugation.

Depending on the intended purpose, the active fusion protein recoveredabove may be used directly, or subjected to further purification bycommonly used purification means, such as ion-exchange chromatography,as needed.

Further, if a site which is cleaved specifically by an enzyme isinserted between the heterologous protein and Cter (or its fragment),treatment of the fusion protein with the enzyme to cleave it at thesequence, followed by removal of the Cter portion (or its fragment),will give the protein of interest.

In order to produce it in the form of a fusion protein in host cells,the molecular weight of a heterologous protein fused with a Cter (or itsfragment) of a Cry protein is preferably not more than 50 kDa.

In a convenient way, detection of a fusion protein obtained by themethod according to the present invention can be done by detecting theCter portion of the fusion protein using an anti-Cter antiserum (or ananti-Cter antibody isolated from the antiserum) collected from an animal(in particular, mammal, e.g., rabbit, goat, etc.) which has beenimmunized with the Cter used in the fusion protein formation accordingto a conventional method (Example 8). For detection, any method wellknown to those skilled in the art may be employed, such as Westernblotting, ELISA, immunoprecipitation, and the like. A column prepared byimmobilizing an antiserum to the Cter or an anti-Cter antibody on asolid phase may also be used for isolation and purification of a fusionprotein.

Further, an antiserum reactive to the original protein before fusion canalso be obtained by immunizing an animal in a conventional manner withthe fusion protein prepared according to the present invention, andcollecting the serum (see Example 7). Such an antiserum or an antibody(polyclonal) to the original protein isolated from the antiserum, can beused in the analysis (detection, semiquantitative or quantitative) of asample (e.g., a biological sample of an animal, esp. of a mammal, amongothers, human tissues, blood, plasma and serum). Specifically, forexample, such an antiserum or antibody is immobilized on a propercarrier, like as latex particles, gelatin particles, colloidal gold,polystyrene beads, or a polystyrene plate. And any of techniques wellknown to those skilled in the art, such as immunoagglutination, enzymelabeling, chemiluminescence, and the like, can be performed on them fordetection of the presence of the original protein contained in a sample,or for semiquantitative or quantitative analysis through comparison witha reference standard. Thus, an antiserum to the original protein (or anantibody to the original protein) obtained from an animal immunized witha fusion protein can be provided as a testing reagent, directly in theform of a solution or a lyophilized preparation, or in such forms inwhich it is immobilized on a proper carrier, like latex particles,gelatin particles, colloidal gold, polystyrene beads, a polystyreneplate, and the like.

Isolation and purification of the antibody to the original protein fromthe antiserum to the fusion protein can be done, in a conventionalmanner, i.e., by preparing an affinity column to which the originalprotein or the Cter employed in the fusion is bound, and by allowing theantibody to the original protein or the antibody to the Cter,respectively, to be specifically adsorbed.

EXAMPLES

The present invention is described in further detail below. It should benoted, however, that the present invention is not intended to be limitedto the examples.

Example 1 Production of Fusion Protein of 4AaCter(696-851) andGlutathione-S-transferase

According to the following procedure, glutathione-S-transferase (GST)originating from Schistosoma japonicum was expressed in a large amountand was let accumulate in E. coli cells, in the form of a fusion proteinwith 4AaCter (696-851), which is a fragment of the Cter of one of Cryproteins, Cry4Aa2, and corresponds to amino acids Ile 698˜Pro 851 ofCry4Aa2.

1. Preparation of a DNA Coding for 4AaCter(696-851) (FIG. 4)

Cry4Aa-S2 gene (formally named, in the priority document, syn4A gene)(which is a gene designed to express a polypeptide consisting of thefull-length Cry4Aa's 1180 amino acids) was synthesized by recursive PCR.For this total synthesis, 50 to 55-base synthetic oligonucleotideprimers were used which had been designed to cover the full-lengthnucleotide sequence of interest and at the same time to form base pairsconsisting of 10 to 15 bases overlapping between adjacent ones of theseprimers. The nucleotide sequence of the open reading frame (ORF) ofcry4Aa-S2 gene is shown as SEQ ID NO:26. Using this as a template, PCRwas carried out to amplify the DNA fragment coding for 4AaCter(696-851).The nucleotide sequences of the primers employed are shown in FIG. 5 andbelow. The DNA fragment obtained by this has a XhoI site at its eachend.

(1) Primer X-Syn4A-C1-f: 5′-GGCTCGAGATCATCAACACCTTCTAC-3′ [nucleotides3-26 (single-underscored in the upper part of FIG. 5) give an XhoI site,nucleotide 9-26 (double-underscored) a terminal sequence of4AaCter(696-851)] (SEQ ID NO:27).

(2) Primer X-S-Syn4A-C1-r: 5′-GGCTCGAGCCCGGGCCGGCACATTCATGATT-3′[nucleotides 3-8 (single-underscored in the lower part of FIG. 5) givean XhoI site, nucleotide 17-31 (double-underscored) a terminal sequenceof 4AaCter(696-851)] (SEQ ID NO:28).

<Reaction Solution>

10 × PCR buffer (for KOD plus) 5.0 μL 2 mM dNTP 5.0 μL 25 mM MgSO₄ 2.4μL Primer X-Syn4A-C1-f (10 μM) 1.5 μL Primer X-S-Syn4A-C1-r (10 μM) 1.5μL Template DNA(25 ng) 1.0 μL Sterilized water (DDW) 32.6 μL  DNApolymerase (KOD plus, TOYOBO) 1.0 μL Total volume 50.0 μL 

<Reaction Conditions>

The above reaction solution was set on a thermal cycler (Gene Amp PCRsystem 9700, PE Applied Biosystems), and reaction was allowed to proceedunder the following condition: 94° C. for 2 min; (94° C. for 15 sec,then 55° C. for 30 sec, then 72° C. for 1 min)×25 cycles; 72° C. for 7min; 4° C. for an indefinite period.

3. Construction of Expression Vector pGST-4AaCter

The fragment obtained above was treated with XhoI. A commerciallyavailable expression vector pGEX-6P-1 (GE Healthcare Bio-Science, FIG.6) was provided. The nucleotide sequence of the multicloning region ofthis vector is shown as SEQ ID NO:29, and the amino acid sequence codedfor by this region as SEQ IN DO:30. Into the Xhol site within the regionwas inserted in a conventional manner the above-mentioned4AaCter(696-851) fragment that had been amplified and then treated withXhol, and thus expression vector pGST-4AaCter was constructed (FIG. 4).This fragment 4AaCter(696-851) is designed in such a manner that GST and4AaCter(696-851) are combined in-frame if it is inserted in the correctorientation. The orientation of the inserted 4AaCter(696-851) gene wasconfirmed based on the restriction enzyme pattern utilizing unique KpnIand NaeI sites within the sequence, and by sequencing.

4. Transformation of Host E. coli Cells by Introduction of the Gene

E. coli BL21 strain cells were transformed by introduction into them ofpGST-4AaCter constructed above. Namely, 0.1 mL of overnight culture ofE. coli BL21 cells was applied to 5 mL of LB medium, and the mixture wasshake cultured at 37° C. until the turbidity of the culture reached 0.5(for about 2 hours). From 1 mL of this, bacterial cells were collectedby centrifugation, suspended in 0.5 mL of ice-cooled 50 mM CaCl₂, andwere let stand on ice for 30 minutes. To a 0.2-mL suspension taken fromthis was added pGST-4AaCter, and the mixture was let stand on ice for 30minutes, then subjected to a heat shock at 42° C. for 30 seconds, and tothis was added 0.8 mL of LB medium (to 1 mL in total). After shakecultured at 37° C. for 1 hour, the culture was streaked ontoampicillin-containing LB agar plates, and after an overnight culture at37° C., an E. coli strain transformed with pGST-4AaCter was obtained.

5. Induction of Expression

The E. coli was precultured. Five mL of the medium (TB) was put in atest tube and cultured overnight. Two mL of this overnight culture wasadded to 200 mL of TB medium. The mixture was cultured on a shakeculture apparatus (New Brunswick Scientific INOVA4230) at 240 rpm for2-3 hours at 37° C., until the OD600 reached 0.6-0.8. IPTG(isopropyl-6-D-thiogalactopyranoside) was added to the culture fluid tomake a final concentration of 0.06 mM. Culture was continued for further2-4 hours (240 rpm 37° C.) to induce expression. Crystal formationwithin the cells was observed (FIG. 7, arrowheads).

6. Solubilization of the Crystals

E. coli cells expressing GST-4AaCter were collected and suspended in 25mL of PBS, and then lysozyme (final concentration: 1 mg/mL) andphenylmethylsulfonyl fluoride (PMSF, final concentration: 1 mg/mL) wereadded to the suspension. The cells then were fractured byultrasonication (for 6 min in total, repeating ON (20 sec) and OFF (10sec)), and insoluble fraction was precipitated by centrifugation at11000 rpm for 15 minutes. The precipitate, which contained crystals ofGST-4AaCter, was washed by centrifugation with a proper volume of PBS,and the precipitate was suspended in 100 mM Na₂CO₃ (pH 10.5) solutionand incubated at room temperature for 1-2 hours to solubilize theGST-4AaCter crystals. After centrifugation, the supernatant, whichcontained GST-4AaCter, was subjected to analysis by SDS-PAGE. It wasconfirmed that the fusion protein was localized in the insolublefraction and solubilized by alkali treatment (FIG. 8).

The GST activity of GST-4AaCter which had been solubilized in a bufferwith a pH of 10.5 was assayed by CDNB (1-chloro-2,4-dinitrobenzene)method. Namely, a protein sample containing GST was put in the wells ofa 96-well plate, and after addition of 200 μL of the following substratesolution to this, let stand for 1 minute at room temperature. Thissample was set on an absorptiometer (Spectra MAX 250, MolecularDevices), and the change in its absorption per unit time was measured at340 nm.

<Substrate Solution (for 4 Samples)>

100 mM potassium phosphate [pH 7.4] 960 μL  50 mM GSH* 20 μL 50 mM CDNB20 μL Total volume  1 mL *GSH: reduced glutathione

The result confirmed that GST-4AaCter exhibits a potent GST activity,though having fallen short of GST as a control (produced from E. colicarrying GST expression vector pGEX-6P-1) (Table 3). Besides, themaximum yield of GST-4AaCter so far reached is 0.6 mg per 3 mL culture.

TABLE 3 GST Activity of Fusion Protein GST activity Protein(μmol/min/nmol GST) GST (control, 27 kDa) 242 GST-4AaCter (fusionprotein, 44 kDa) 181

Example 2 Production of Fusion Protein of Various Fragments of Cry4A andGlutathione-S-Transferase

Study was made to identify where the indispensable region resides in4AaCter for a fusion protein to form crystals. As shown in FIG. 9,various parts of the amino acid sequence Cry4Aa were amplified, andtheir fusion proteins with GST were produced according to a proceduresimilar to that followed in Example 1. Each of them was examined fortheir formation of crystals in the same manner as in Example 1. Thefusion protein with 4AaCter(852-1180) was not found to form clearcrystals, nor did the fusion protein with GST-4AaCter(696-799) exhibitformation of crystals. On the other hand, crystal formation wasconfirmed with GST-4AaCter(696-851) [amino acid sequence of theCter(696-851) portion: SEQ ID NO:6] and with those fusion proteinsprepared using gradually shortened peptide chains, i.e.,GST-4AaCter(801-851), GST-4AaCter(801-834) [amino acid sequence of the4AaCter(801-834) portion: SEQ ID NO:31], and GST-4AaCter(801-829) [aminoacid sequence of the 4AaCter(801-829) portion: SEQ ID NO:7]. Thisindicates that in 4AaCter, the sequence essential for crystal formationis a polypeptide chain portion consisting of 29 amino acids, 801-829,equally included in these latter Cter portions.

Example 3 Production of Fusion Protein of 4AaCter(696-851) with MM29 kD

MM29 kD, a cytotoxic protein derived from B. thuringiensis, exhibits apotent toxicity to mammalian cells (esp. to leukemia and cancer cells).MM29 kD is produced and accumulated in B. thuringiensis cells in theform of a precursor consisting of 304 amino acids (whose nucleotide andamino acid sequences shown as SEQ ID NO:32 and SEQ ID NO:33,respectively), and converted to the active form through removal of 28amino acids from the N-terminus and some from the C-terminus byproteinase K. Though it is not known as yet how many amino acids areremoved from its C-terminus in nature, removal of 23 amino acids givesan active protein. And based on studies of its molecular size, it isconsidered that about 23 amino acids are removed also in nature. Thenucleotide sequence of the active MM29 kD, which is formed by removal ofN-terminal 28 amino acids and C-terminal 23 amino acids, is presented asSEQ ID NO:34, and the corresponding amino acid sequence as SEQ ID NO:35.

While it has so far been prepared through its expression in the form ofa fusion protein with GST in E. coli cells, it has been difficult toobtain MM29 kD in high yield. Namely, MM29 kD is a protein which cannotbe expressed easily. This is thought to be due to the adverse effects ofMM29 kD, which accumulates in the soluble fraction, on the viability andreproduction of the E. coli cells. An attempt was made to let thisprotein accumulate in the form of crystals (i.e., in a manner that thesoluble fraction is kept free of this protein) and thus obtain highyield while averting the adverse effects. For this purpose, fusionproteins were constructed in which 4AaCter was added in-frame to eitherthe N- or C-terminus of MM29 kD, respectively, i.e., 4AaCter-MM29 kD andMM29 kD-4AaCter (FIG. 10).

1. Construction of Expression Vector for Production of Fusion Protein4AaCter-MM29 kD (FIG. 11)

Using the total DNA from B. thuringiensis MM50G2 strain as a templateand the following set of primers, PCR was performed to prepare the MM29kD gene DNA having a BamHI site on the upstream end and an EcoRI site onthe downstream end (FIG. 10).

(1) Primer Cytox-N-f-Bam: (SEQ ID NO: 36)GTGGATCCGTTATTCAAGAATACCTTACGTTTAATG (2) Primer Cytox-r-999-Eco:(SEQ ID NO: 37) AGGAATTCAAGCTTCTTGCTGTTCAGC

A commercially available protein expression vector pGEX-4T-3 wasprovided (FIG. 12). The nucleotide sequence of the multicloning regionof this expression vector is presented as SEQ ID NO:38, and the aminoacid sequence coded for by this as SEQ ID NO:39. One day mutagenesisusing PCR was applied in a conventional manner to remove from the vectorthe entire open reading frame (ORF) of GST leaving behind its startcodon “ATG” alone to prepare pΔGST-4T-3 (FIG. 11). The nucleotidesequence of pΔGST-4T-3, part of which is shown in FIG. 13, is presentedas SEQ ID NO:40. And the forward primer and the reverse primer shown inFIG. 13 employed in this process are presented as SEQ ID NO:41 and SEQID NO:42, respectively. Using these primers, BamHI was insertedimmediately after that start codon “ATG” left in pΔGST-4T-3.

Then, the above DNA fragment (which has a BamHI site on the upstream endand an EcoRI site on the downstream end) was inserted in a conventionalmanner into the BamHI/EcoRI site of pΔGST-4T-3 to form vector pΔGST-MM29kD. A region of nucleotide sequence near the start codon “ATG” of GSTand 5′-end of the above-mentioned DNA coding for MM29 kD is given inFIG. 14 and as SEQ ID NO:43.

The DNA fragment (BamHI)-4AaCter-(BamHI) was inserted into the BamHIsite of pΔGST-MM29 kD prepared above to construct vectorpΔGST-4AaCter-(MM29 kD).

Then, using this as a template, one day mutagenesis was performed toreplace the nucleotide sequence “GCGGATCC”, which connected between4AaCter and MM29 kD, with “GCG”. 4AaCter and MM29 kD thus were connectedin-frame and gave pΔGST-4AaCter-MM29 kD (FIG. 11).

2. Construction of Expression Vector for Production of Fusion ProteinMM29 kD-4AaCter (FIG. 15)

Using PCR, DNA fragment (BamHI)-MM29 kD-(HindIII) was prepared by addinga nucleotide sequence “GGATCC” to the 5′ end of the DNA sequence codingfor MM29 kD (active form) and a nucleotide “TGAATTC” to the 3′ end,respectively. This process added to the 3′ end of MM29 kD a stop codon“TGA”, and provided a EcoRI site simultaneously. At the same time, aHindIII site was freshly created immediately upstream of this EcoRIsite.

A PCR was performed using the DNA coding for 4AaCter. For this process,nucleotide sequences of the primers had been designed so that HindIIIrestriction sites might be added at either end of this DNA, and thatin-frame connection to the terminal HindIII site might become possible(namely, so that “AAGCTTTA” might be added to the 5′ end of the DNAcoding for 4AaCter, and “AAGCTT” at the 3′ end).

A recombinant vector pBSII-MM29 kD was prepared by inserting the aboveDNA fragment, (BamHI)-MM29 kD-(HindIII), into the BamHI/HindIII site ofa commercially available cloning vector pBluescript II SK+ (pBSII,Stratagene, USA).

Then, by inserting (HindIII)-4AaCter-(HindIII) fragment into the HindIIIsite of the recombinant vector pBSII-MM29 kD (thus MM29 kD and 4AaCterlinked in-frame), pBSII-MM29 kD-4AaCter was constructed.

By treating pBSII-MM29 kD-4AaCter with a BamHI/XhoI restriction enzyme,a BamHI/XhoI DNA fragment including the (BamHI)-MM29kD-4AaCter-(HindIII) region was cut out, which then was inserted intothe BamHI/XhoI site of pGEX-4T-3 to form pGST-MM29 kD-4AaCter.

One-day mutagenesis using PCR was applied using pGST-MM29 kD-4AaCter DNAas a template, the ORF of GST was removed up to the BamHI recognitionsite sequence leaving the first “ATG” (start codon) alone, and thetac-promoter upstream of GST and the start codon “ATG” was directlylinked in-frame to the MM29 kD gene, to form pΔGST-MM29 kD-4AaCter (FIG.15).

3. Transformation of Host E. coli Cells by Introduction of a Gene andCollection of Crystals

E. coli BL21 cells were transformed by introduction either of thepΔGST-4AaCter-MM29 kD or of the pΔGST-MM29 kD-4AaCter, which had beenconstructed above 1 and 2, respectively. Five mL of TB medium wasinoculated with either of the transformant BL21 strain cells, and afteraddition of 10 μL of 50 mg/mL Amp, shake cultured at 37° C. for 12 hours(preculture). Fifty-mL TB medium was inoculated with 500 μL of thepreculture, and 100 μL of 50 mg/mL Amp was added to this. Shake culturewas done at 37° C. for 3 hours (OD₆₀₀=about 0.8). To this was added 50μL of 100 mM IPTG, and culture was continued at 37° C. for 3 hours.Formation of crystals was confirmed in the E. coli cells transformedeither with 4AaCter-MM29 kD or with MM29 kD-4AaCter (FIG. 16,arrowheads). The bacterial cells were collected and suspended in 20 mLof PBS [pH7.5] and centrifuged (10000 rpm, 4° C., 10 min: RS-18IV/TOMY). The precipitate separated was suspended in ice-cooledsterilized water, then fractured by ultrasonication (for 5 min in total,repeating ON (10 sec) and OFF (10 sec)), and centrifuged (10000 rpm, 4°C., 10 min). The precipitate, after washed by centrifugation (not lessthan 3 times) with ice-cooled sterilized water, was suspended in 2 mL of50 mM Tris-HCl [pH 7.4]. This suspension was subjected to sucrosedensity-gradient centrifugation, and the layer which contained crystals(white band) was collected and suspended in 20 mL of PBS [pH7.5]. Thiswas centrifuged (12000 rpm, 4° C., 10 min) and the precipitate wasfurther centrifuged in ice-cooled sterilized water (at least 3 times).The supernatant was fully removed, and the precipitate was suspended in20 mL of ice-cooled sterilized water. After additional centrifugation(12000 rpm, 4° C., 10 min), precipitate was suspended in 2 mL ofice-cooled sterilized water, then distributed into sample tubes, andstored at −80° C.

4. Solubilization of Crystals

The precipitate was suspended in a solubilization buffer (100 mM Na₂CO₃[pH 10.5], 10 mM DTTz) to solubilize the crystals (37° C., 30 min)(crystals can be easily solubilized and collected at pH 10-12). Aftercentrifugation (14000 rpm, 4° C., 10 min), the supernatant (4AaCter-MM29kD of MM29 kD-4AaCter) was collected. This was separately purified byanionic column chromatography (HiTrap Q XL, GE Healthcare Bioscience).The proteins adsorbed by the column and then was eluted using 100 mM,200 mM, and 300 mM NaCl stepwise. 4AaCter-MM29 kD and MM29 kD-4AaCterwere found to be eluted with 300 mM NaCl. Examination using SDS-PAGErevealed that these standard samples had been purified to a single band(FIG. 17).

5. Release and Collection of MM29 kD from Fusion Protein

Then, to the supernatant containing 4AaCter-MM29 kD or MM29 kD-4AaCterwas added proteinase K in a proper amount ( 1/10 of the amount of theprotein in the supernatant) to remove the 4AaCter sequence upstream ordownstream of MM29 kD (37° C., 1 hour). MM29 kD, which is ratherresistant to decomposition with proteinase K, survives this treatment asthe core polypeptide. The reaction was terminated by 0.1 M PMSF(phenylmethylsulfonium fluoride) which was added to a finalconcentration of 1 mM. The protein was purified again by anionicion-exchanger chromatography. MM29 kD is not adsorbed by the column andflows through. Analysis of the purified MM29 kD by SDS-PAGE confirmedthat either protein was purified to a single band.

6. Assessment of Cytotoxicity

Using the proteins, 4AaCter-MM29 kD, MM29 kD-4AaCter, and free MM29 kD,purified above, assessment was made for cytotoxicity (lethal activity)of the purified fusion proteins by MTT assay employing Jurkat cells,which originate from a leukemia cell, as a target. Briefly, the numberof the Jurkat cells (purchased from Institute of Physical and ChemicalResearch) were counted using Burker Turk Deep ( 1/10 mm). The cells werediluted to the density of 5.0×10⁵ cells/mL with a medium for measurementfor assay (RPMI 1640 free of phenol red, Nissui). The cell culture wasplaced, 90 μL each, in a necessary number of wells of a 96-well plate.Ten μL each of the samples which had been diluted to properconcentrations with PBS [pH7.5] was added to each well, and incubationwas made (37° C., 3 hrs). Five mg/mL MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma,dissolved in the medium for measurement) then was added to each well,and incubation was made (37° C., 3 hrs). 100 μL of acidic isopropanolwas added to each well, and after sufficient pipetting, OD (570 nm) wasmeasured by a spectrophotometer to calculate survival rate of the cells.As a result, 4AaCter-MM29 kD exhibited an activity comparable toGST-MM29 kD, which had been used as the standard sample in the studiesof MM29 kD. Compared to these, the activity of MM29 kD-4AaCter was foundto be several times higher (Table 4). Free MM29 kD, prepared either from4AaCter-MM29 kD or MM29 kD-4AaCter, exhibited its EC50 at 0.4-0.5 ng/mL(Table 4), which suggests that it is not that MM29 kD is irreversiblyinactivated when it is joined by 4AaCter at its N-terminus, but probablyis lowered in its apparent biological activity due to some sterichindrance. Besides, the yield of 4AaCter-MM29 kD and MM29 kD-4AaCterobtained above was 0.3-0.5 mg per 50 mL of culture, which was at least 6times the yield of GST-MM29 kD (usually, about 50 μg per 50 mL ofculture), which had so far been utilized to produce MM29 kD in E. colias a host.

TABLE 4 Cytotoxicity of Fusion Protein to Jurkat Cells ProteinCytotoxicity (EC50, ng/ml) GST-MM29kD 23.4 4AaCter-MM29kD 24.6MM29kD-4AaCter 4.7 Free MM29kD 0.4-0.5

Example 4 Production of Fusion Protein of Cter of Cry1Aa andGlutathione-S-Transferase

As it was confirmed as shown in Examples 1-3 that GST and MM29 kD can beproduced in a large amount using E. coli in the form of their fusionproteins with various fragments of 4AaCter, an attempt then was made toproduce fusion proteins with the Cter of other Cry proteins. Using1AaCter, the Cter of Cry1Aa (insecticidal toxin specific to lepidopteraninsects (butterflies and moths)), which exhibits an insecticidalspectrum utterly different from that of Cry4Aa (insecticidal toxinspecific to dipteran insects (mosquitoes)), a large-scale expression ofGST was tried as described below.

1. Preparation of a Gene Coding for GST-1AaCter(622-777) andConstruction of an Expression Vector pGST-1AaCter

Based on the result of alignment [using a software ClustalW] with theamino acid sequence of 4AaCter(696-851), a corresponding peptide,1AaCter(622-777), was selected. To prepare a fusion protein with thispeptide chain, a gene fragment containing the coding region for1AaCter(622-777) (amino acid sequence set forth as SEQ ID NO:1) wasamplified by PCR. This PCR was performed using DNAs extracted from B.thuringiensis subsp. sotto T84A1 strain as a template, together with thefollowing primers, which are specific to 1AaCter:

(1) Primer 1Aa3-C1-f: (SEQ ID NO: 44) GGATCCGCGGTGAATGAGCTG(2) Primer 1Aa3-C1-r: (SEQ ID NO: 45) CTCGAGACCCACATTTACTGT

The gene fragment (nucleotide sequence set forth as SEQ ID NO:46) whichwas thus amplified is provided with a BamHI site at its upstream end anda XhoI site at its downstream end, respectively. This fragment wasinserted in-frame into the BamHI-XhoI site within the multicloning sitewhich is downstream of the GST gene of the expression vector pGEX-6P-1,giving an expression vector, pGST-1AaCter (FIG. 18). pGST-1AaCter isdesigned to express the fusion protein of GST and 1AaCter (GST-1AaCter).

2. Transformation of E. coli Host by Introduction of the Gene

E. coli BL21 cells were used as the host. The cells were transformed byintroduction of the above expression vector pGST-1AaCter in the samemanner as in Examples 1 and 2, and following induction of expression,GST-1AaCter was expressed. Thus, formation of crystals was observed inthe cells, as was the case with GST-4AaCter (FIG. 19, arrowheads).SDS-PAGE analysis of the supernatant (soluble protein fraction) and theprecipitate (insoluble protein fraction) separated by centrifugationshowed that GST-1AaCter, which was estimated to be about 45 kDa, waslocated mainly in the insoluble protein fraction (FIG. 20). The amountof expressed GST-1AcCter was estimated to be about 5 μg per 10 μg of E.coli total proteins, according to computer image analysis. While theGST-1AaCter crystals were scarcely soluble in alkaline buffer solutionswith a pH of 9-11, they were solubilized in a buffer with a pH of 12(FIG. 21). Measurement of GST activity was made by the CDNB assay andrevealed their high activity comparable to that of the GST purifiedstandard [purified from pGEX-6P-1-introduced E. coli cells throughGlutathione Sepharose 4B (GE Healthcare)] (Table 5).

TABLE 5 GST activity of GST-1AaCter GST activity Protein (μmol/min/nmolGST) GST purified standard 107.3 GST- 1 AaCter 99.8

Example 5 Preparation of Fusion Protein with Cter from Cry1Ac andGlutathione-S-Transferase

Cry1Ac is an insecticidal toxin specific to lepidopteran insects(butterflies and moths) like Cry1Aa employed in Example 4. Cry1Ac,however, exhibits different characteristics from those of Cry1Aa, suchas its N-acetylgalactosamine (GalNAc) recognizing lectin activity. Anexamination was carried out in the following manner to find whether aprotein (GST) linked to the Cter (1AcCter) from this Cry1Ac proteinwould form crystals and accumulate in E. coli cells.

1. Preparation of Gene Coding for GST-1AcCter(624-779) and Constructionof Expression Vector pGST-1AcCter

Based on the result of alignment [using a software ClustalW] with theamino acid sequence of 4AaCter(696-851), a corresponding part,1AcCter(624-779), was selected from the sequence of Cry1Ac1. In order toprepare a fusion protein with this, a gene fragment containing thecoding region for 1AcCter(624-779) (amino acid sequence set forth as SEQID NO:4) was amplified by PCR. This PCR was performed using DNAsextracted from B. thuringiensis subsp. kurstaki HD73 strain as atemplate, together with the following primers, which are specific to1AcCter(624-779):

(1) Primer 1Ac1-C1-f: (SEQ ID NO: 47) GGATCCGCGGTGAATGCGCTG(2) Primer 1Ac1-C1-r: (SEQ ID NO: 48) CTCGAGTGGCACATTTACTGT 

The gene fragment (nucleotide sequence set forth as SEQ ID NO:49) whichwas thus amplified was provided with a BamHI site at its upstream endand a XhoI site at its downstream end, respectively. This fragment wasinserted in-frame into the BamHI-XhoI site downstream of the GST gene ofthe expression vector pGEX-6P-1, which gave pGST-1AcCter an expressionvector for a fusion protein of GST and 1AcCter (GST-1AcCter).

2. Transformation of E. coli host by Introduction of the Gene

Using E. coli BL21 cells in the same manner as in Example 1, CST-1AcCterwas expressed. As a result, formation of crystals was observed in thecells, as were the cases with GST-4AaCter and GST-1AaCter (FIG. 22,arrowheads). SDS-PAGE analysis of the supernatant (soluble proteinfraction) and the precipitate (insoluble protein fraction) separated bycentrifugation showed that GST-1AcCter, which was estimated to be ofabout 45 kDa, was found localized mainly in the insoluble proteinfraction (FIG. 23). The amount of expressed GST-1AcCter was about 5 μgper 10 μg of the E. coli total proteins, as estimated by computer imageanalysis. While the GST-1AcCter crystals were scarcely soluble inalkaline solutions with a pH of 9-11, they were solubilized in a bufferwith a pH 12 (FIG. 24). Measurement of the activity of GST-1AcCter thussolubilized was made by the CDNB assay and revealed that its activitywas comparable to that of the purified standard (Table 6).

TABLE 6 GST activity of GST-1AcCter GST activity Protein (μmol/min/nmolGST) GST purified standard 107 GST-1AcCter 105

Example 6 Preparation of Fusion Protein with Cter from Cry8Ca

An attempt then was made to prepare a fusion protein with a Cter fromCry8Ca1. Based on the result of alignment [using a software ClustalW]with the amino acid sequence of 4AaCter(696-851), a correspondingsequence, 8CaCter(672-829), was selected. In order to prepare a fusionprotein with this, a vector having a DNA in which a gene fragment codingfor 8CaCter(Lys672-Pro829) (amino acid sequence set forth as SEQ IDNO:14) had been inserted in-frame downstream of a gene fragment codingfor GST was introduced to E. coli BL21 strain cells to transform these,and the expression of the fusion protein was induced. As a result,formation of crystals was observed in the E. coli cells.

Example 7 Preparation of C-Reactive Protein (CRP) Utilizing 4AaCter

C-reactive protein (CRP), which occurs in the blood in response to aninflammation, is an inflammation marker, and its measurements in theblood: which is performed using an antibody reactive specifically to CRP(anti-CRP antibody), can be used as an index to the activity andseverity, in follow-up observations and prognosis of an inflammatorydiseases. There is a high demand for anti-CRP antibody, and so is thedemand for CRP itself, which is the necessary immunogen in order toproduce the antibody. However, as CRP collected and purified from thebody contains a substantial amount of contaminants originating from itssources, an anti-CRP antibody prepared using it as the immunogen couldreact with other compounds occurring in the body, thereby affecting themeasurement. For this reason, as well as for steady supply of theimmunogen CRP, it has been desired that production of anti-CRP antibodyis made using, as the immunogen, a recombinant CRP produced bymicroorganisms. Thus, an attempt was made to produce CRP utilizing4AaCter.

<Construction of Expression Vector for Production of Fusion Protein>

Human CRP gene which had been prepared by gene synthesis in thefollowing manner was inserted in-frame between the BamHI and XhoI sitesof the above mentioned pΔGST-4T-3 to construct pΔGST-CRP.

Namely, the gene segment encoding human CRP was synthesized by recursivePCR performed with reference to a database (GenBank NM_(—)00567). PCRwas performed using a DNA fragment prepared by PCR using a pair ofprimers each of which had at an end a sequence which was complementaryto the sequence at an end of the other: i.e., CRP_(—)1f (SEQ ID NO:97)(6nucleotides at the 5′-end of this form a BamHI site) and CRP_(—)2r (SEQID NO:98); and fragments prepared by PCR using a series of primers eachhaving at its ends sequences complementary to its flanking primers:i.e., CRP_(—)3f (SEQ ID NO:99), CRP_(—)4r (SEQ ID NO:100), CRP_(—)5f(SEQ ID NO:101), CRP_(—)6f (SEQ ID NO:102), CRP_(—)7f (SEQ ID NO:103),CRP_(—)8r (SEQ ID NO:104), CRP_(—)9f (SEQ ID NO:105), CRP_(—)10r (SEQ IDNO:106), CRP_(—)11f (SEQ ID NO:107), CRP_(—)12r (SEQ ID NO:108)(6nucleotides at the 5′-end of this form a XhoI site), and thus a DNAcoding for the full length CRP (SEQ ID NO:109)(respective 6 nucleotidesat 5′- and 3′-ends form restriction sites required in subcloning) wasprepared. In the amino acid sequence (SEQ ID NO:110) coded for by theDNA, the parts consisting of two amino acids at the N- and C-termini,respectively, are linker sequences which have been brought in as part ofthe restriction sites. The DNA (SEQ ID NO:109) coding for the fulllength human CRP was inserted in-frame into pΔGST-4T-3 which had beendigested with BamHI and XhoI, to construct pΔGST-CRP.

Then a fragment coding for 4AaCter was inserted in-frame into the BamHIsite of pΔGST-CRP to construct pΔGST-4AaCter-CRP. Namely, PCR wasperformed using the open reading frame of the above-mentioned cry4Aa-S2gene as a template, and using primers B-Syn4A-C1-f (SEQ ID NO:111)(6nucleotides at its 5′-end form a BamHI site) and B-Syn4A-C1-rn (SEQ IDNO:112)(6 nucleotides at its 5′-end form a BamHI site) to amplify theDNA fragment coding for 4AaCter(696-851). The fragment thus obtained isprovided with BamHI sites at it both ends. The reaction solution andreaction conditions for this PCR were as follows.

<Reaction Solution>

10 × PCR buffer (for KOL plus) 5.0 μL 2 mM dNTP 5.0 μL 25 mM MgSO₄ 2.4μL Primer B-Syn4A-C1-f (10 μM) 1.5 μL Primer B-Syn4A-C1-rn (10 μM) 1.5μL Template DNA (25 ng) 1.0 μL Purified water (DDW) 32.6 μL  DNApolymerase (KOD plus, TOYOBO) 1.0 μL Total volume 50.0 μL 

<Reaction Condition>

The above reaction solution was set in a thermal cycler (Gene Amp PCRsystem 9700, PE Applied Biosystems) and reaction was allowed to proceedunder the following condition: 94° C. for 2 min; (94° C. for 15 sec,then 55° C. for 30 sec, then 72° C. for 1 min)×25 cycles; 72° C. for 7min; 4° C. for an indefinite period.

4AaCter(696-851) thus prepared was inserted into a plasmid obtained bydigestion of pΔGST-CRP with BamHI to create pΔGST-4AaCter-CRP.pΔGST-4AaCter-CRP will express a fusion protein of 4AaCter and CRP(4AaCter-CRP).

<Expression of Fusion Protein>

Thus created pΔGST-4AaCter-CRP was introduced into E. coli BL21 straincells. The procedures of introduction of this expression vector andinduction of expression of E. coli cells were the same as thosedescribed above with regard to the introduction of pGST-4AaCter into E.coli cells and induction of its expression. Expression of 4AaCter-CRPwas confirmed as follows: the cells were collected and suspended in 10mL PBS, and after subjected to sonication (ON for 20 sec, OFF for 10sec), the buffer containing the fractured cells was run in SDS-PAGEtogether with human CRP (which had been obtained by inserting human CRPgene into a BamHI and XhoI-digested plasmid pΔGST-4T-3, and introducingthe plasmid thus obtained into E. coli cells and inducing itsexpression). As a result, while a band of interest was detected at about48 kDa with CRP fused to 4AaCter in comparison with the run of a sampletaken before induction of expression, no band of interest was observedat about 29 kDa with CRP which had been expressed without fusion with4AaCter (FIG. 25).

Western blotting of this and human native CRP biological sample usinganti-4AaCter-CRP goat antiserum confirmed that this antiserum wasreactive to both 4AaCter-CRP and native CRP (FIG. 26). The above resultsthus indicate that expression of CRP is now available by fusing 4AaCterwith CRP, and that an antibody reactive to native CRP can be obtainedusing 4AaCter-CRP as the immunogen.

<Method for Preparation of Anti-4AaCter-CRP Goat Antiserum)

A goat which had been kept for at least one week for habituation wasimmunized with 2 mg of 4AaCter-CRP mixed with Freund's completeadjuvant, 5 times at 2-week intervals, and blood was taken from thejugular vein. The blood thus obtained was kept at 37° C. for one hourand let stand at 4° C. for a day and a night. The supernatant obtainedwas centrifuged at 3000 rpm for 5 minutes, and the supernatant thusobtained was used as 4AaCter-CRP antiserum.

Example 8 Preparation of Anti-4AaCter Antiserum

Immunoassays using an antibody are often performed following expressionprocesses of recombinant proteins, as a method to confirm whether theproteins expressed are the intended ones. However, it is time consumingand costly to provide antigens which are specific to the proteins ofinterest. For this reason, a method is employed in which a protein ofinterest is expressed in the form a fusion protein with some other knownprotein and the expression of the protein of interest is confirmed usingan antibody specific to the known protein. Thus, an examination wascarried out to ascertain whether protein expression can be confirmedusing an anti-4AaCter antibody in the case of a 4AaCter fusion protein.

pΔGST-4AaCter was created by inserting in-frame a gene fragment codingfor 4AaCter into the BamHI site of pΔGST, which had been made byremoving the GST gene from pGEX4T-3. This vector, pΔGST-4AaCter,expresses 4AaCter in E. coli cells.

4AaCter was let express in E. coli BL21 cells, and after the cells werefractured, the precipitate that was fractionated by centrifugation wassolubilized in an alkaline buffer with a pH of 12. Rabbits wereimmunized in a conventional manner with the solubilized 4AaCter, andanti-4AaCter antiserum was obtained. Namely, rabbits (New Zealand White)which had been kept for one week for habituation were immunized with 0.5mg of 4AaCter mixed with Freund's complete adjuvant 5 times at two-weekintervals, and blood was taken from the jugular vein. The blood thusobtained was kept at 37° C. for one hour and let stand at 4° C. for aday and a night. The supernatant thus obtained was centrifuged at 3000rpm for 5 minutes, and the supernatant thus obtained was used as 4AaCterantiserum.

Western blotting of 4AaCter and 4AaCter-CRP using the 4AaCter antiserumobtained above confirmed that this antiserum was reactive to both4AaCter and 4AaCter-CRP (FIG. 27). This result indicates that it ispossible to generate an antiserum reactive to 4AaCter by using 4AaCteras the immunogen, and that the 4AaCter antiserum is also reactive to the4AaCter fusion protein.

INDUSTRIAL APPLICABILITY

The present invention enables to produce a heterologous protein as afusion protein with a Cter, in bacterial cells, such as E. coli cells,in a great amount in the form of insoluble crystals retaining theprotein's activity, which crystals can be solubilized and recovered asan active protein. Thus, the present invention is utilized forproduction of heterologous proteins using bacteria, such as E. coli, asa host. Further, an antiserum which is created using a fusion protein ofthe present invention can be utilized in analysis of the originalprotein, the protein before fusion with a Cter, e.g., as an testingreagent.

[Sequence Listing]

GP124-PCT.ST25

1. A method for production of a protein (A) in the form of a fusionprotein, comprising the steps of (a) preparing a DNA which codes for afusion protein comprising the peptide chain forming the protein (A) andother peptide chain (B), on the N- or C-terminal side of the latter theformer being combined, wherein the peptide chain (B) is a C-terminalpeptide chain included in the amino acid sequence of one of the Cryproteins produced by Bacillus thuringiensis listed in Table 1 or 2 andincluding in itself a corresponding partial sequence identified in thetables, TABLE 1 C-terminal peptide, and Cry proteins partial sequence(SEQ ID NO:) Cry1Aa1(SEQ ID NO: 50), Cry1Aa2(SEQ ID NO: 51), C-terminalpeptide: the part starting Cry1Aa3(SEQ ID NO: 52), Cry1Aa4(SEQ ID NO:53), from Ala622 Cry1Aa5(SEQ ID NO: 54), Cry1Aa8(SEQ ID NO: 55), Partialsequence: Ala622-Pro777 Cry1Aa9(SEQ ID NO: 56), Cry1Aa10(SEQ ID NO: 57),(SEQ ID NO: 1) Cry1Aa11(SEQ ID NO: 58), Cry1Aa12(SEQ ID NO: 59),Cry1Aa13(SEQ ID NO: 60), Cry1Aa14(SEQ ID NO: 61) Cry1Ab3(SEQ ID NO: 62),Cry1Ab4(SEQ ID NO: 63), C-terminal peptide: the part startingCry1Ab8(SEQ ID NO: 64), Cry1Ab9(SEQ ID NO: 65), from Ala623 Cry1Ab10(SEQID NO: 66), Cry1Ab12(SEQ ID NO: 67), Partial sequence: Ala623-Pro778Cry1Ab13(SEQ ID NO: 68), Cry1Ab15(SEQ ID NO: 69), (SEQ ID NO: 2)Cry1Ab16(SEQ ID NO: 70), Cry1Ab17(SEQ ID NO: 71), Cry1Ab21(SEQ ID NO:72) Cry1Ab2(SEQ ID NO: 73) C-terminal peptide: the part starting fromAla624 Partial sequence: Ala624-Pro779 (SEQ ID NO: 3) Cry1Ac1(SEQ ID NO:74), Cry1Ac4(SEQ ID NO: 75), C-terminal peptide: the part startingCry1Ac7(SEQ ID NO: 76), Cry1Ac8(SEQ ID NO: 77), from Ala624 Cry1Ac9(SEQID NO: 78), Cry1Ac10(SEQ ID NO: 79), Partial sequence: Ala624-Pro779Cry1Ac11(SEQ ID NO: 80), Cry1Ac16(SEQ ID NO: 81), (SEQ ID NO: 4)Cry1Ac19(SEQ ID NO: 82) Cry1Ac5(SEQ ID NO: 83), Cry1Ac12(SEQ ID NO: 84),C-terminal peptide: the part starting Cry1Ac14(SEQ ID NO: 85),Cry1Ac15(SEQ ID NO: 86), from Ala623 Cry1Ac20(SEQ ID NO: 87) Partialsequence: Ala623-Pro778 (SEQ ID NO: 5)

TABLE 2 C-terminal peptide, and Cry proteins Partial sequence(SEQ IDNO:) Cry4Aa1(SEQ ID NO: 88), C -terminal peptide: the part Cry4Aa2(SEQID NO: 89), starting from Ile696 Cry4Aa3(SEQ ID NO: 90) Partialsequence: Ile696-Pro851 (SEQ ID NO: 6 or Ile801-Ser829 (SEQ ID NO: 7)Cry4Ba1(SEQ ID NO: 91), C-terminal peptide: the part Cry4Ba2(SEQ ID NO:92), starting from Val652 Cry4Ba5(SEQ ID NO: 93) Partial sequence:Val652-Pro807 (SEQ ID NO: 8) or Ile757-Ser785 (SEQ ID NO: 9) Cry4Ba4(SEQID NO: 94) C-terminal peptide: the part starting from Val651 Partialsequence: Val651-Pro806 (SEQ ID NO: 10) or Ile756-Ser784 (SEQ ID NO: 11)Cry4Ba3(SEQ ID NO: 95) C-terminal peptide: the part starting from Val652Partial sequence: Val652-Pro807 (SEQ ID NO: 12) or Ile756-Ser784 (SEQ IDNO: 13) Cry8Ca1(SEQ ID NO: 96) C-terminal peptide: the part startingfrom Lys672 Partial sequence: Lys672-Pro829 (SEQ ID NO: 14)

(b) introducing the DNA into a host bacterium to transform the same, and(c) allowing the fusion protein to be expressed in the host bacteriumwhich has been transformed.
 2. The method for production according toclaim 1, wherein in the step of preparing the DNA which codes for afusion protein comprising the peptide chain forming the protein (A) andthe other peptide chain (B), on the N- or C-terminal side of the latterthe former being combined, a DNA coding for an amino acid sequence whichprovides a specific cleavage site for a proteolytic enzyme is interposedbetween the DNA which codes for the peptide chain forming protein (A)and the DNA which codes for the peptide chain (B).
 3. The method forproduction according to claim 1 comprising a further step of fracturingthe host bacterium containing the fusion protein thus expressed tocollect the fusion protein.
 4. The method for production according toclaim 3 comprising a further step of purifying the collected fusionprotein through solubilization of the same in an alkaline aqueoussolution.
 5. The method for production of the protein (A) comprising astep of removing the peptide chain (B) from the fusion protein obtainedaccording to claim
 4. 6. The method for production according to claim 5,wherein the removal of the peptide chain (B) is done by treating aspecific cleavage site for a proteolytic enzyme, which the fusionprotein has between the peptide chain forming the protein (A) andpeptide chain (B), with the proteolytic enzyme.
 7. A fusion proteinproduced by the method according to claim
 3. 8. An antiserum reactive tothe protein (A) which antiserum is obtained by immunizing a mammaliananimal with the fusion protein according to 7 above, and then collectingthe serum of the animal.
 9. The antiserum according to claim 7, whereinthe protein (A) is C-reactive protein.
 10. An antibody to the protein(A) isolated from the antiserum according to claim
 8. 11. A testingreagent comprising the antiserum according to claim
 8. 12. A testingreagent comprising the antibody according to claim
 10. 13. A method foranalysis of a sample for protein (A), comprising the steps of bringingthe sample into contact with the antiserum according to claim 8 above,to form an antigen-antibody complex, and detecting the antigen-antibodycomplex.
 14. The method according to claim 13, wherein the protein (A)is C-reactive protein.